The aim of this project is to examine the mechanism of gene expression of the negative-strand viruses. Experiments will be performed using vesicular stomatitis virus (VSV). These experiments will be designed to distinguish between the two proposed models for transcription. One model suggests that each viral message is separately initiated and terminated while the other proposes that a polycistronic RNA is made and processed into monocistronic messages. Three types of polycistronic RNA are synthesized by VSV and/or by one of its defective particles. The structure and biosynthesis of each of the three classes will be examined to determine if any of these RNAs serves as a precursor to monocistronic message. The RNAs will be physically characterized by electron microscopy and by nucleic acid hydridizations. Each will be tested as a substrate for nucleolytic processing by virion-associated endonucleases. Sequencing of relevant regions of the viral genome will shed light on the roles of specific sequences in the regulation of gene expression. Methylation appears to be directly involved in the control of VSV gene expression. The methylases present in the VSV-infected cell will be isolated by affinity chromatography. The proteins will be characterized by polyacrylamide gel electrophoresis and methylation assays will be performed to identify the substrates.